Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π–π and π–cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S–Se–Se chemical moiety.


S4
. Analytical HPLC analysis of compound 31 (conditions II) Figure S3. Analytical HPLC analysis of compound 32 (conditions II) S5 Figure S4. Analytical HPLC analysis of compound 33 (conditions I)  S2. Enzymatic studies. The native urease of S. pasteurii CCM 2056 was purified using a five-step chromatographic procedure, as previously described. [S10]  (1-60 mM) using the phenol-hypochlorite method, as previously reported. [S11] One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 µM ammonia per min under specific conditions.

S2.1. Inhibition studies. Competitive reversible inhibition.
Inhibition studies (compounds 30-36, 38-41, 44-51, 53 and 54) were carried out by initiating the enzyme reaction with the addition of 119.3 pM S. pasteurii urease in assay mixtures (200 μL total volume) containing increasing concentrations of inhibitors and 1−60 mM of urea. The values were calculated using the appropriate equations in the GraphPad Prism 5 software. The inhibition mechanism was determined using Lineweaver-Burk plots after testing at least five inhibitor concentrations in the range depending on their inhibitory strength (exemplified in Figure S26).
of the reaction product formed at time t, which is the reaction time, and are the reaction initial and steady-state rates, respectively, and is the apparent first-order rate constant for the interconversion between and ). The control curves in the absence of inhibitor were linear. The equilibrium dissociation constants of the initial and the final enzyme conformation complex * ( and , respectively) were determined using equation: is the Michaelis-Menten constant, maximum rate of enzyme in a noninhibited reaction, S and I are substrate and inhibitor concentration). The mechanism of inhibitor binding was determined using steady-state kinetic measurements ( Figures S27 and S28). The enzyme (final concentration 119.3 pM) was pre-incubated with increasing inhibitor concentrations in separate aliquots of reaction buffer prior to the initiation of the reaction by urea incorporation at a concentration ranging from 1 to 60 mM. The incubation time was different for the assayed inhibitors, depending on when equilibrium was achieved between enzyme , inhibitor and enzyme-inhibitor complexes ( and : . The values were calculated using the appropriate equations implemented in + ↔ ↔ * ) the GraphPad Prism 5 software. The inhibitors were dissolved in water or DMF (the solvent was found to be non-inhibitory up to a concentration of 20%). Urease activity was determined using the Berthelot colorimetric reaction as previously described. [S11,S12] The determinations of values were run in triplicate for the compounds analyzed. artificial urine was used in which urea was omitted, sodium citrate was replaced with 0.5% glucose S20 and 0.01% phenol red added. [S13] The assays were carried out in tubes containing 2.0 mL of modified urine and various concentrations of compounds tested. Cells were induced to ureolysis and introduced into the reaction mixture in the proportion described above. After 1.5 hours of preincubation with gentle mixing at 25 °C, sterile urea solution (30 mM final concentration) was added and the urease reaction was carried out for another 1.5 hours. 120 µL samples were withdrawn every 10 min and briefly centrifuged to remove precipitate that can occur in artificial urine medium due to elevation of the pH induced by urease. The supernatant (100 µL) was placed in a microplate well and immediately read at 570 nm.
The IC 50 and IC 90 parameters expressed the specific inhibitor concentration at which the increase in absorbance per minute was 50% and 10% of the rate of the untreated control, respectively. The results were calculated from three independent assays using seven inhibitor concentrations.

S3.1. Proteus mirabilis PCM543 viability control.
The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] was obtained from Sigma-Aldrich. 10 μL of MTT solution (1 mg mL -1 in 10 mM PBS, pH 7.2) was added to 90 μL of urease reaction samples collected at the end of each assay, which contained cells exposed to the compounds tested for a total of 3 hours. After 1 hour of incubation at 25 °C, 100 µL of acidic isopropanol (1.5% (v/v) solution of hydrochloric acid in isopropanol) was added. MTT formazan was allowed to solubilize for 15 min and the absorbance was read at 550 nm.
S4. Molecular modeling. The crystal structure of S. pasteurii urease with 1.50 Å resolution (PDB id 5G4H) was used as the starting point for the calculations. [S14] The structure was prepared for the calculations using the Discovery Studio Visualizer v. 4.1 (BIOVIA): (a) hydrogen atoms were automatically added assuming a pH of 7.0, (b) the protonation of amino acid residues that form active sites was manually checked and adjusted, and (c) the partial charges of all atoms were assigned using the Momany-Rone algorithm. Minimization of the inhibitor-enzyme covalent complex (e.g. Figure   S21 S29) was performed using the CHARMM program. [S15] Smart Minimizer algorithm and the CHARMM force field were used for this purpose. Minimization was performed up to an energy change of 0.0 or an RMS gradient of 0.1. Residues that did not form the active site cleft were frozen.
No implicit solvent model was applied. The non-bond radius was set to 14 Å. Diffraction data were collected at 100 K using synchrotron X-ray radiation at the EMBL P13 beamline of the Petra III storage ring, c/o DESY, Hamburg (Germany). [S16] Helical data collection was performed to achieve higher data quality by minimizing protein crystal radiation damage. Data processing and reduction were carried out using XDS [S17] and AIMLESS. [S18,S19] The crystals belonged to space group P6 3 22, isomorphous with all the crystal structures of S. pasteurii urease determined so far. The initial phases for the structure determination were obtained using the X-ray crystal structure of S. pasteurii urease bound to catechol (PDB code 5G4H, 1.50 Å resolution) [S14] as a phasing model, devoid of solvent molecules, catechol and other ligands, and following coordinates randomization in order to remove any potential phase bias. Structural determination was conducted by restrained refinement with REFMAC5. [S20] Model rebuilding, as well as water or ligand addition/inspection, were manually conducted using COOT. [S21,S22] Unbiased omit electron density maps for non-proteinaceous ligands were calculated using Fourier coefficients F o -F c and phases from the last cycle of restrained refinement before the addition of the ligands in the refining model.

S23
The X-ray crystal structure was refined using isotropic atomic displacement parameters (ADPs) (including the hydrogen atoms in the riding positions), at a final resolution of 1.54 Å, and deposited in the Protein Data Bank with the accession code 7ZCY. Data collection, processing and final refinement statistics are given in Table S1. Selected distances and angles around the Ni(II) ions in the present structure were compared with the corresponding data obtained for the crystal structure of native S. pasteurii urease (PDB code 4CEU, Table S2). The active site and mobile flap regions of both structures are superimposed in Figure S30.  (Table S3). , where I is the intensity of a reflection, and is the mean intensity of all symmetry related

S24
reflections j; c , where I is the intensity of a reflection, and is the mean intensity of all symmetry related reflections j, and N is the multiplicity; d Taken from REFMAC; [S20] R free is calculated using 5% of the total reflections that were randomly selected and excluded from refinement; e , where N atoms is the number of the atoms included in the refinement, N refl is the number of the reflections included in the refinement, D max is the maximum resolution of reflections included in the refinement, compl is the completeness of the observed data, and for isotropic refinement, N params  4N atoms . [S23] S25   Figure S30. (a) Close-up of mobile flap and active site regions of S. pasteurii urease bound to the di-nuclear Se cluster, superimposed to the same environment of the crystal structure of the native enzyme (PDB code 4CEU). [S24] The mobile flaps are represented as ribbons and colored light blue and transparent green for the Se-bound and the native structures, respectively. Side chains of the selected residues are shown as stick, where carbon, nitrogen, oxygen, and sulfur atoms are grey, blue, red and yellow, respectively (transparent representation for those belonging to the native enzyme). The Ni and Se atoms are shown as green and orange spheres,